Protein Extraction From Cowpea Tissues for 2-D Gel Electrophoresis and MS Analysis E ´ . A. R. Vasconcelos, F. C. S. Nogueira, E. F. M. Abreu, E. F. Gonc ¸alves, P. A. S. Souza, F. A. P. Campos & Department of Biochemistry and Molecular Biology, Universidade Federal do Ceara ´ , 600001-970, Fortaleza Ceara ´, Brazil; E-Mail: bioplant@ufc.br Received: 21 March 2005 / Revised: 14 July and August 2005 / Accepted: 15 August 2005 Online publication: 10 October 2005 Abstract A method of extraction of proteins from cowpea for two-dimensional electrophoresis is pre- sented. This method avoids loss of protein in the course of sample preparation and results in a fraction that yields reproducible 2-DE protein patterns. The efficiency of this method was demonstrated by comparison of the patterns of protein deposition in developing and mature cowpea seeds. It is also demonstrated that without further processing of the gel piece the proteins present in the spots can be directly digested with trypsin and the peptides subjected to mass spectrometry analysis for identification. Keywords Two-dimensional electrophoresis Mass spectrometry analysis Protein extraction Cowpea proteins Vigna unguiculata Introduction The tissues from the different plant spe- cies present a wide diversity and concen- tration of secondary metabolites, carbohydrates and lipids. This has pre- vented the development of methods for protein extraction and analysis by 2D- electrophoresis that can be applied to plant tissues in general [1–3]. For exam- ple, besides the presence of phenolic and other components of the secondary metabolism, the seeds of legumes present a very high concentration of high molec- ular weight carbohydrates, that cause a decrease in the amount of protein ex- tracted [4, 5]; additionally carbohydrates as well as other nonprotein contaminants remaining in the protein fractions, affect adversely the IEF in the first dimension as well as the resolution of the 2D gels [1]. We show here a protocol for the extrac- tion of total proteins from mature seeds and embryogenic cell suspensions of cowpea (Vigna unguiculata), an important legume crop in many areas world wide. We demonstrated the efficiency of this protocol by comparing protein expression during zygotic embryogenesis in cowpea at four stages of seed development. Additionally we have identified two pro- teins from cell suspensions by PMF and one protein from zygotic embryos by MS– MS sequencing. Experimental Plant Material Developing seeds and embryogenic cell suspensions of cowpea (Vigna unguiculata L. Walp) were chosen for this study. Cowpea seeds at different developmental stages were obtained as described by Carasco and Xavier-Filho [6]. Embryo- genic cell suspensions were obtained by cultivating mature embryo axis of cow- pea in complete MS medium [7], supple- mented with 2,4-dichlorophenoxyacetic acid (2,4-D) at 1.0 mg L )1 , glutamine at 100 mg L )1 and casein hydrolysis at 100 mg L )1 , solidified with 0.7% agar, in the dark at 27Æ2°C After three subculti- vation at intervals of thirty days, a cell suspension was obtained by transferring five grams of calli into a 250 mL Erlen- meyer flasks containing 30 mL of the induction medium. Protein Extraction In a typical experiment, the plant mate- rial is freeze-dried and a fine powder is obtained by grinding in a coffee mill and sieving through a 100 mesh metal sieve. Protein extraction is performed by mixing the powder (0,2 g), insoluble polyvinyl- pyrrolidone and extraction buffer (50 mM pyridine, 10 mM thiourea, 1% SDS, pH 5.0) in the proportion 1:2:40 (w/ w/v). The mixture is stirred for two hours at 4°C and centrifuged at 10,000 g for 40 min. The supernatant is mixed with 4 2005, 62, 447–450 DOI: 10.1365/s10337-005-0637-1 0009-5893/05/10 Ó 2005 Friedr. Vieweg & Sohn/GWV Fachverlage GmbH Short Communication Chromatographia 2005, 62, October (No. 7/8) 447